Development of Potent and Selective Antagonists ... - ACS Publications

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Development of Potent and Selective Antagonists for the UTPActivated P2Y4 Receptor Muhammad Rafehi,†,§ Enas M. Malik,†,§ Alexander Neumann,† Aliaa Abdelrahman,† Theodor Hanck,† Vigneshwaran Namasivayam,† Christa E. Müller,*,† and Younis Baqi*,‡ †

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany ‡ Department of Chemistry, Faculty of Science, Sultan Qaboos University, PO Box 36, Postal Code 123, Muscat, Oman S Supporting Information *

ABSTRACT: P2Y4 is a Gq protein-coupled receptor activated by uridine-5′-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y4 receptor antagonist has been described so far. Therefore, we developed and optimized P2Y4 receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescencebased assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4dimethylphenylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, 61) exhibited an IC50 value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand−receptor interactions. Compound 64 (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most selective P2Y4 receptor antagonist known to date. Compounds 61 and 64 are therefore anticipated to become useful tools for studying this scarcely investigated receptor.

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demonstrated to mediate chloride (Cl−) secretion in the jejunal epithelium and therefore represent possible targets for the treatment of cystic fibrosis (P2Y4 receptor agonists) and diarrhea (P2Y4 receptor antagonists).13−16 P2Y4 receptors also appear to be involved in the regulation of amyloid precursor protein (APP) production and its release in the brain.17 Because APP accumulation is associated with the progression of Alzheimer’s disease, P2Y4 receptor antagonists might be useful as therapeutic agents for this debilitating and yet incurable disorder.17 In the heart, P2Y4 receptors are expressed on cardiac endothelial cells; they were found to be important for cardiac endothelial cell growth and migration as well as secretion of platelet-derived growth factor B (PDGF-B). Moreover, P2Y4 receptor-knockout mice showed defective postnatal cardiac development, including reduced heart weight. It has thus been postulated that P2Y4 receptors could be possible targets to modulate angiogenesis and regulate cardiac remodeling and postischemic revascularization.18 In the cochlea, P2Y4 receptors

INTRODUCTION The G protein-coupled P2Y receptors represent, together with the ATP-gated ion channels known as P2X receptors, the nucleotide-activated P2 receptor family. Eight subtypes of P2Y receptors are known to exist: P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 belonging to the P2Y1-like subgroup, and P2Y12, P2Y13, and P2Y14 that are members of the P2Y12-like subgroup of P2Y receptors.1,2 P2Y receptors are present in almost all human tissues, where they exert various biological functions. They are of great interest as (potential) therapeutic targets for several indications, including neurodegenerative disorders, pain, cancer, and cardiovascular diseases.3,4 In fact, several antithrombotic drugs on the market, namely clopidogrel, prasugrel, and ticagrelor, achieve their effect through inhibiting the human platelet P2Y12 receptor.5−7 Despite a generally strong interest in the P2Y receptor family, relatively little is known regarding the P2Y4 subtype. This uridine-5′-triphosphate (UTP)-activated receptor shows a wide distribution in the body, including heart, gastrointestinal tract (GIT), central nervous system (CNS), skin, and ear (cochlea).8−12 In the GIT, P2Y4 receptors were © 2017 American Chemical Society

Received: January 12, 2017 Published: March 17, 2017 3020

DOI: 10.1021/acs.jmedchem.7b00030 J. Med. Chem. 2017, 60, 3020−3038

Journal of Medicinal Chemistry

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Figure 1. Structures of the classical P2 receptor antagonists PPADS, Reactive Blue 2 (RB-2), suramin, and receptor subtype-selective tool compounds derived from RB-2.

antagonists of purine receptors and ectonucleotidases.24,27−36 For example, we have previously developed PSB-0739 (4, Figure 1),30 an extremely potent and selective competitive P2Y12 receptor antagonist (pA2 of 9.8),37 and PSB-1011 (5, Figure 1), a competitive inhibitor of the rat P2X2 receptor with a Ki value of 79 nM.31 High potency and selectivity could be attained, for example, by optimizing the residue at the 4-position of the anthraquinone core. Structure−activity relationships (SARs) were found to be very different at the different purine receptor subtypes, and the anthraquinone core structure can thus be described as a “privileged structure” in medicinal chemistry.26 The aim of this study was to specifically design and develop the first potent and selective P2Y4 receptor antagonist. To this end, a library of RB-2-related anthraquinone derivatives was synthesized using recently developed methods,27−29,39 and the compounds were tested for their potency to block P2Y4 receptors using a Ca2+-mobilization assay.59 This data served as a basis for subsequent structure−activity relationship analyses and receptor docking studies aimed at enhancing our knowledge with respect to P2Y4 receptor ligand preferences and for further optimizing the structures. Modifications of the substituents at the 4-position of the anthraquinone core led to

are expressed in the epithelial cells of Reissner’s membrane, where they regulate Na+ absorption upon noise exposure,8 and in the apical membrane, where they are involved in K+ secretion across strial marginal cell epithelium during stimulation of the cochlea by sound.9 So far, no selective antagonists for the P2Y4 receptor have been described.1 However, such a compound is required as a pharmacological tool for receptor characterization and for studies aimed at target validation.19 Mouse and rat P2Y4 receptors, which share 51% and 83% sequence identity with the human P2Y4 receptor,12,20 are noncompetitively antagonized by PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, 1, Figure 1) and also blocked by Reactive Blue 2 (RB-2, 2, Figure 1).21,22 Rat P2Y4 receptors, expressed in Xenopus oocytes, were found to be weakly antagonized by suramin (3, Figure 1).23 On the other hand, human P2Y4 receptors expressed in 1321N1 cells were ineffectively inhibited by PPADS and were insensitive to suramin23 but were inhibited by RB-2 at micromolar concentrations.24 RB-2 is a well-known nonselective P2 receptor antagonist that has served as an important pharmacological tool in the field of purinergic signaling.25,26 Our laboratory has been extensively working on the development of anthraquinone derivatives structurally related to RB-2 as potent and selective 3021



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Table 1. Yields, Molecular Weights, UV Absorption, Color, and Purity of the Newly Synthesized Anthraquinone Derivatives

compd Structure 14 19 20 21 24 25 33 34 35 37 39 40 41 42 43 44 49 52 53 67 Structure 58 59 60 61 62 63 64 Structure 68 69 a

R1 A SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na CH3 B SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na SO3Na C SO3H SO3H

yield (%)a

MW (g/mol)

absorption λmax (nm)

color

purity by LC-MS/UV (%)b

2-chloro 2-methyl 3-methyl 4-methyl 4-ethyl 3-propyl 2,3-dimethyl 2,4-dimethyl 2,5-dimethyl 3,4-dimethoxy 3-methoxy-4-methyl 4-chloro-2-methyl 4-chloro-3-methyl 4-hydroxy-3-methyl 4-fluoro-3-methoxy 4-chloro-3-methoxy 2-carboxy-3-fluoro 2-carboxy-4-hydroxy 2-carboxy-4-nitro 2-carboxy-4-fluoro

36 80 70 72 64 35 49 60 50 46 44 36 40 40 43 40 40 68 21 21

450.83 430.41 430.41 430.41 444.44 458.46 444.44 444.44 444.44 476.43 460.43 464.85 464.85 446.41 464.40 480.85 478.38 476.39 505.39 390.36

590 624 626 626 626 628 624 624 624 624 626 626 624 626 624 624 594 630 584 612

blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue

99.0 99.0 99.1 100.0 99.0 97.7 99.0 99.0 99.5 98.5 98.3 99.0 99.0 96.0 99.3 99.7 98.0 100.0 95.7 99.7

4-fluorophenoxy 4-chlorophenoxy 4-bromophenoxy 2,4-dimethylphenylthio 2,5-dimethylphenylthio 3,4-dimethylphenylthio 3-pyridylmethylthio

41 25 17 10 7 7 14

526.47 542.92 587.38 552.59 552.59 552.59 539.56

630 630 625 640 640 640 640

blue blue blue blue blue blue blue

99.5 98.6 96.1 96.1 98.0 95.9 98.6

fluoro methoxy

97 38

397.38 409.41

500 506

purple purple

98.5 95.2

R2 (A,C) or R2-X (B)

Isolated yield. bPurity of the compounds was determined using LC-MS coupled to a diode array detector (220−900 nm).

an increase in potency at the P2Y4 receptor and resulted in selectivity for P2Y4 over other P2Y receptor subtypes.

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Scheme 1. General Synthesis of 4-Substituted Anthraquinone Derivatives (7−53 and 67)a

RESULTS AND DISCUSSION

Chemistry. The target compounds were synthesized as depicted in Schemes 1−3. The syntheses of several compounds had been previously described.24,27−30,33,36,38−40 Some of the known anthraquinone derivatives (19−21, 24, and 33−35 (Table 1)) have now been obtained by improved synthetic procedures. In addition to known compounds, a series of 21 new compounds was prepared. Condensation of bromaminic acid (R1 = SO3H (6a)) or 1-amino-4-bromo-2-methylanthraquinone (R1 = CH3 (6b)) with the appropriate amine yielded the target compounds in satisfactory to excellent isolated yields (Scheme 1). Reactions were conducted in phosphate buffer (pH 6−7) in the presence of copper powder (Cu0) under microwave irradiation at 80−120 °C.

a

Reagents and conditions: (i) R2-NH2, phosphate buffer (pH 6−7), Cu0, microwave, 80−120 °C, 5−24 min.

Compounds 65 and 66 were synthesized from 1-amino-4bromo-2-methylanthraquinone (6b) by treatment with an excess of the appropriate aniline derivative (15 equiv) under argon in the presence of potassium acetate and a catalytic amount of copper(I) acetate at 110 °C for 6−15 h (Scheme 2). 3022



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Scheme 2. Synthesis of Anthraquinone Derivatives 65 and 66a

a

Reagents and conditions: (i) CuOAc, KOAc, 110 °C, argon, 6−15 h.

according to the following principle: monoclonal colonies of 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor were loaded with the acetoxymethyl ester of the calcium-sensitive fluorescent dye fluo-4. The P2Y4 receptor is coupled to Gq protein. Thus, receptor activation with the endogenous ligand UTP will lead to an increase in the intracellular calcium ion concentration and a rise in the intensity of fluorescent light emitted by the dye. Test compounds that act as P2Y4 receptor antagonists inhibit the increase in fluorescence intensity, which is monitored using a microplate fluorescence reader. Prior to evaluating the antagonistic activity of the anthraquinone derivatives, a concentration−response curve of the agonist UTP at the human P2Y4 receptor was obtained (Figure 2). The EC80 of

Three anilinoanthraquinone derivatives, 12, 27, and 46, which bear a primary amino group in the 1-position of the anthraquinone moiety, were treated with sodium nitrite in hydrochloric acid solution (1 M) at 0 °C for 5 min, then allowed to warm up to room temperature followed by the addition of ethanol and an excess of zinc powder (10 equiv) to achieve deamination,39 affording the desired products 68−70 (Scheme 3). Scheme 3. Synthesis of Deaminated Anthraquinone Derivatives 68−70a

a Reagents and conditions: (i) NaNO2, HCl (1 M), 0−5 °C, 5 min; (ii) Zn (10 equiv), ethanol, rt, 30 s.

Purification of Reactive Blue 2. RB-2 has been used for decades as a valuable pharmacological tool for studying nucleotide receptors. Most studies relied on commercially available products. However, there has always been some doubt regarding the identity and purity of commercial products, making it difficult to obtain reliable pharmacological results.41−43 RB-2 is a mixture of two isomers bearing a sulfonate group either in position 3 or 4 of ring F (compound 2, Figure 1). Thus, we decided to investigate the purity of commercially available RB-2. The purity of the investigated RB2 was declared to be 88.2% as determined by HPLC-UV. However, the employed technique is not suitable to detect inorganic impurities, e.g., inorganic salts. Therefore, we purified commercially available RB-2 by reversed phase-18 flash column chromatography (RP-18 FCC, for details see Experimental Section). Subsequent analysis showed that the commercial product only contained 54% of RB-2, while the main organic contaminant was found to be the precursor of RB-2, namely disodium 1-amino-4-[4-(4,6-dichloro-[1,3,5]triazin-2-ylamino)3-sulfonatophenylamino]-9,10-dioxo-9,10-dihydroanthracene2-sulfonate (ca. 12%). Moreover, it was found to be contaminated with ca. 34% of inorganic salts, presumably NaCl and Na3PO4, which were washed out during the purification step using RP-18 column chromatography. Biological Assays. The potencies of the compounds were determined using a fluorescence-based Ca2+-mobilization assay

Figure 2. Concentration−response curve of the agonist UTP determined by calcium mobilization assays on recombinant 1321N1 astrocytoma cells stably expressing the human P2Y4 receptor. Data points shown are the mean values of eight independent experiments, each performed in duplicate. The determined EC50 of UTP is 203 ± 41 nM. The calculated EC80 value is 565 nM.

UTP at this cell line was determined to be 565 nM. A UTP concentration of 500 nM, close to its EC80 value, was subsequently used for receptor activation for the determination of antagonist potency using the calcium mobilization assay. A total of 64 synthesized anthraquinone derivatives including 21 new compounds not previously described in the literature along with the commercially obtained and the purified RB-2 were tested for their inhibitory activity at the human P2Y4 receptor. The IC50 values are summarized in Tables 2−4. Structure−Activity Relationships. Purified RB-2 (2′) displayed an IC50 value of 0.625 μM and hence was more potent than the commercially available RB-2 (IC50 = 1.14 μM, Figure 3 and Table 2). This is very reasonable, because the dye content of commercially available RB-2 was found to be only 54%. While the Hill slope of the concentration−inhibition curve for purified RB-2 (2′) was found to be close to unity, 3023



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These results suggested that a phenylamino substituent at the 4-position of the anthraquinone core structure is preferred for P2Y4 receptor inhibitory potency over a benzyl or phenethyl residue. Therefore, a library consisting of 60 derivatives of lead structure 7 with various mono- and di-substitutions on the phenyl ring attached to the 4-amino group was investigated for inhibitory potency at the P2Y4 receptor (see Table 3). Introducing different lipophilic substituents in the ortho-, meta-, and para-positions in ring D, e.g. a halogen atom (F, Cl, Br), an alkyl (CH3, C2H5, C3H7), or a methoxy group, did not alter the activity, which ranged between 1 and 9 μM (compounds 11−28, Table 3). On the other hand, introducing hydrophilic moieties led to a reduction in potency by over a 10fold in the case of meta- and para-carboxy, while ortho-carboxy and para-hydroxy substitutions were tolerated (compounds 29−32, Table 3). In the next step, we assessed di-substitution in ring D (Table 3): di-substitution with lipophilic residues did not alter the compounds’ potency (compounds 33−41 and 43−45, Table 3). However, a combination of lipophilic and hydrophilic substituents affected the activity in three different directions: 3methyl-4-hydroxy (42; IC50 = 0.746) enhanced the antagonistic activity by over 4-fold, 2-carboxy-5-chloro (51; IC50 = 2.76) was tolerated, whereas any other combination, 2-carboxy-4fluoro (46), 2-carboxy-4-chloro (47), 2-carboxy-4-bromo (48), 2-carboxy-3-fluoro (49), 2-carboxy-5-fluoro (50), 2-carboxy-3hydroxy (52), and 2-carboxy-3-nitro (53), dramatically decreased the inhibitory activity up to over a hundred-fold (Table 3). To investigate the effect of the presence of an additional ring E, 11 new compounds (54−64) were synthesized. In the first step, different bridges (CH2, O, NH, S) in the para-position of ring D were investigated. All four linkers were tolerated (compounds 54−57, IC50 = 3.45, 1.94, 1.91, and 1.18 μM, respectively, Table 3). In the next step, different substituents were introduced into ring E, including halogen (F, Cl, Br) and methyl groups. 4-Fluorophenoxy and 4-bromophenoxy residues were found to have no influence on the antagonistic activity (compounds 58 and 60, IC50 = 1.69 and 1.76, respectively, Table 3), while 4-chlorophenoxy substitution increased the potency by >8-fold (59, IC50 = 0.373). Di-methylation in ring E with a sulfide linker between ring D and E improved potency by more than 13-fold (compounds 61−64, IC50 = 0.233, 0.395, 0.482, and 0.409 μM, respectively, Table 3 and Figure 4). Indeed, compound 61 (IC50 = 0.233 μM) can be considered as the most potent P2Y4 receptor antagonist known to date. As a next step, we were interested in investigating the effects of other substituents on the anthraquinone core, namely the amino group in the 1-position and the sulfonate group in the 2position. Therefore, we developed six new analogues (65−70, Table 4) of selected anthraquinone derivatives (12, 27, and 46). No improvement in the activity of compound 46 (IC50 > 100, Table 3) was observed upon replacement of the sulfonate group with a methyl group (compound 67) or upon deamination at the 1-position (compound 70). Moreover, replacement of the 2-sulfonate group of compounds 12 and 27 (IC50 = 1.39 and 1.70 μM, respectively, Table 3) with a methyl group completely abolished the activity (compounds 65 and 66, respectively, Table 4). Similar results were obtained with the deaminated compounds 68 and 69 (Table 4). These results indicate that the presence of an amino group in the 1-position and a sulfonate function in the 2-position of the anthraquinone core are essential for antagonistic activity at the P2Y4 receptor.

Figure 3. Concentration−response curves of the commercially available RB-2 (2) and purified RB-2 (2′), determined using the calcium-mobilization assay on recombinant 1321N1 astrocytoma cells stably expressing the human P2Y4 receptor. UTP at a concentration of 500 nM (∼EC80) was used for receptor activation. Data points shown are the mean values of at least three independent experiments, each performed in duplicate. The IC50 values are 1.14 ± 0.31 for the commercially available RB-2 (2), Hill slope −2.20, and 0.625 ± 0.198 for the purified RB-2 (2′), Hill slope −0.80.

crude RB-2 had a much steeper Hill slope of −2.20. This might be due to allosteric modulatory effects of the contaminants present in the nonpurified material, e.g., inorganic salts.44 Regarding the synthesized compounds, the structurally simple 1-amino-4-phenylamino-2-sulfoanthraquinone, acid blue-25 (AB-25, 7), showed inhibitory activity at low micromolar concentrations (IC50 = 3.10 μM), being almost as potent as the much larger RB-2 (IC50 = 1.14 μM). Replacing the phenylamino ring by benzylamino, phenethylamino, or αnaphthylamino (8−10) decreased the activity by more than 2fold (Table 2). Table 2. Antagonistic Activity of Anthraquinone Derivatives at the Human P2Y4 Receptor, Part I

a

Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP (EC80) in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. 3024



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Table 3. Antagonistic Activity of Anthraquinone Derivatives at the Human P2Y4 Receptor, Part II

compd

R1

R2

R3

R4

IC50 ± SEM (μM)a (or % inhibition at indicated concentration)

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 4624 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

F H H Cl H H H H CH3 H H C2H5 H H H OMe H H H CO2H H H CH3 CH3 CH3 H H H H CH3 H H H H H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H H H H H H H H H H H

H F H H Cl H Br H H CH3 H H C2H5 H C3H7 H OMe H H H CO2H H CH3 H H CH3 OMe OMe OMe H CH3 CH3 OMe OMe F H H H F H H H H H H H H H H H H H H

H H F H H Cl H Br H H CH3 H H C2H5 H H H OMe OH H H CO2H H CH3 H H OMe H CH3 Cl Cl OH F Cl H F Cl Br H H H OH NO2 benzyl phenoxy phenylamino phenylthio 4-fluorophenoxy 4-chlorophenoxy 4-bromophenoxy 2,4-dimethylphenylthio 2,5-dimethylphenylthio 3,4-dimethylphenylthio

H H H H H H H H H H H H H H H H H H H H H H H H CH3 CH3 H OMe H H H H H H F H H H H F Cl H H H H H H H H H H H H

1.97 ± 0.42 1.39 ± 0.36 4.04 ± 1.01 9.34 ± 2.07 2.34 ± 0.71 3.03 ± 0.66 6.96 ± 0.73 3.30 ± 0.09 3.49 ± 0.29 2.22 ± 0.31 2.82 ± 0.92 3.53 ± 1.21 1.51 ± 0.48 3.21 ± 0.91 2.99 ± 0.12 3.98 ± 1.39 1.70 ± 0.24 2.59 ± 0.72 3.16 ± 0.58 6.10 ± 1.72 23.4 ± 3.6 ≈30 (57%)b 2.63 ± 0.59 3.58 ± 0.25 11.2 ± 1.2 6.45 ± 2.22 4.98 ± 0.99 9.25 ± 4.56 4.13 ± 0.99 2.20 ± 0.52 1.65 ± 0.37 0.746 ± 0.076 1.54 ± 0.66 3.37 ± 1.09 12.5 ± 2.3 >100 (17%)c >100 (31%)c >100 (30%)b >100 (10%)c >100 (33%)c 2.76 ± 0.75 21.1 ± 7.1 >30 (30%)b 3.45 ± 0.41 1.94 ± 0.54 1.91 ± 0.28 1.18 ± 0.09 1.69 ± 0.38 0.373 ± 0.112 1.76 ± 0.49 0.233 ± 0.079 0.395 ± 0.082 0.482 ± 0.137

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Table 3. continued compd

R1

R2

R3

R4

0.409 ± 0.138

see structure above

64


a

Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP (EC80) in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. b% Inhibition at 30 μM. c% Inhibition at 100 μM.

Figure 5. Summary of structure−activity relationships of anthraquinone derivatives as P2Y4 receptor antagonists.

Figure 4. Concentration−response curves of selected anthraquinone derivatives, determined using the calcium mobilization assay with recombinant 1321N1 astrocytoma cells stably expressing the human P2Y4 receptor. UTP at a concentration of 500 nM (EC80) was used for receptor activation. Data points shown are the mean values of at least three independent experiments, each performed in duplicate. The IC50 values are found in Tables 2 and 3.

studies on the SARs of RB-2 and related dyes, which showed that AB-25 is the minimum structural requirement for biological activity of this class of compounds.24,29,30,33,45−52 Moreover, the activity on the P2Y4 receptor was strongly affected by the substituents present on ring E, e.g., a lipophilic substitution in the ortho-, meta-, or para-position increased the activity, while an unsubstituted ring E with an −SCH2− linker was also tolerated. It is interesting to compare the results of the SAR analysis at the human P2Y4 receptor with that at other purinergic targets (Figure 53, Supporting Information). The main difference between the human P2Y4 receptor and both the human P2Y12 receptor and P2Y1-like receptors of the guinea-pig taenia coli (receptor was not cloned but found to be pharmacologically

Comparison of Structure−Activity Relationships of Anthraquinone Derivatives at Different Targets. An overview of the structure−activity relationships (SARs) of the investigated anthraquinone derivatives at the human P2Y4 receptor is depicted in Figure 5. The ABCDE aromatic ring system proved to be important for high inhibitory potency. The amino function in the 1-position and the sulfonate group in the 2-position of the anthraquinone scaffold were found to be essential for the activity. This is in accordance with previous

Table 4. Inhibitory Potencies at the Human P2Y4 Receptor of Selected Anthraquinone Derivatives Modified in the 1- and 2Positions

compd Structure A 12 27 46 Structure B 65 66 67 Structure C 68 69 70

R1

R2

R3


H H CO2H

F OMe H

H H F

1.39 ± 0.36 1.70 ± 0.28 >100 (17%)c

H H CO2H

F OMe H

H H F

>30 (−4%)b >30 (38%)b ≈100 (57%)c

H H CO2H

F OMe H

H H F

≈30 (49%)b >10 (77%)b >100 (25%)c

a

Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP (EC80) in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. bPercent inhibition at 30 μM. cPercent inhibition at 100 μM. 3026



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Table 5. Selectivity of the Most Potent P2Y4 Receptor Antagonists versus Other P2Y Receptor Subtypesa

IC50 ± SEM (μM) or (% inhibition at 50 μM) compd

R

7 42 61 62 63 64

H 3-methyl-4-hydroxy 2,4-dimethyl 2,5-dimethyl 3,4-dimethyl see structure above

hP2Y4 3.10 0.746 0.233 0.395 0.482 0.409

± ± ± ± ± ±

0.35 0.076 0.079 0.082 0.137 0.138

hP2Y1 17.6 7.65 5.48 2.72 4.55 ∼25 (61

± ± ± ± ± ±

hP2Y2 6.5 1.90 0.34 0.59 0.42 4%)

17.1 16.4 8.54 6.52 10.2 13.1

± ± ± ± ± ±

6.3 4.5 1.45 1.04 1.5 2.6

hP2Y6 2.79 4.75 12.5 2.67 4.36 ≫ 100 (12

± ± ± ± ± ±

hP2Y12 0.17 1.49 3.9 0.91 0.60 19%)

3.12 0.0604 2.41 2.96 2.34 3.59

± ± ± ± ± ±

1.17 0.0147 0.45 0.24 0.10 0.38

a

Potency to inhibit calcium mobilization in 1321N1 astrocytoma cells recombinantly expressing human P2Y recetors following receptor activation with 500 nM ADP (P2Y1), 500 nM UTP (P2Y2, P2Y4), or 750 nM UDP (P2Y6). Potency at the Gi-coupled P2Y12 receptor was determined using a β-arrestin translocation assay.

similar to the recombinant P2Y1-receptor subtype)41 is that substitution of ring D with a sulfonate group at the metaposition (for P2Y12 receptors)30,37 and at the ortho-position (for the P2Y1-like receptors) was essential for the inhibitory activity of the compounds. An additional ring F was also important in case of the P2Y1-like receptor.32,42 For activity at the AMP-hydrolyzing enzyme ecto-5′-nucleotidase, ring system ABCD was sufficient for the inhibitory activity.24 In contrast, a triazine or pyrimidine as ring E was preferred by the P2X2 receptor.31 On the other hand, development of anthraquinone compounds as large conductance Ca2+-activated K+ channel openers showed a preference for a bulky and hydrophobic ring D.53 Interestingly, the amino group at ring C was not essential for activity at that target.53 These differences in SARs emphasize the influence of the nature of substituents at the 4-position of the anthraquinone ring on the potency and target selectivity of the compounds, and allow the development of receptor (subtype)-selective ligands. Selectivity. To gain insights into the selectivity of these compounds, we assessed the most potent compounds of this series, 61−64, on the receptor subtypes that share the greatest sequence homology with the P2Y4 receptor, namely P2Y1, P2Y2, and P2Y6, which belong to the subgroup of Gq proteincoupled P2Y1-like P2Y receptors. In addition, we investigated selected compounds at the P2Y12 receptor that had previously been shown to be potently blocked by anthraquinone derivatives.30 Table 5 and Figure 6 summarize the results. Of this series of compounds, 64 is the most selective one, with particularly high selectivity versus the P2Y1 and P2Y6 receptors, where it showed only low potency. Selectivity against the P2Y12 receptor was also observed. It can therefore be concluded that these compounds display P2Y4 receptor selectivity, and it is not only the potency but also the selectivity of lead structure 7 that has been significantly enhanced (see Figure 6). The Binding Mode of Anthraquinone Derivatives at the P2Y4 Receptor. The mode of antagonism, competitive or noncompetitive, at the P2Y4 receptor was determined using the most potent compound (61) of the present series. Concentration−response curves of the endogenous ligand UTP after a preincubation with fixed concentrations of 61 were obtained.

Figure 6. Potencies of selected anthraquinone derivatives on different human P2Y receptors determined using the calcium mobilization assay (P2Y1−6 receptors) or β-arrestin translocation assay (P2Y12 receptor). Shown are the pIC50 values of at least three independent experiments performed in duplicate.

For a competitive antagonist, a parallel rightward shift of the curves toward higher concentrations of UTP would be expected with increasing concentrations of 61. The upper plateaus of the curves, corresponding to the maximum receptor activation, should remain unaltered. A corresponding Schild plot would show a straight line with a slope of approximately 1. Noncompetitive antagonism, on the other hand, is characterized by a suppression of the maximum receptor activation with increasing antagonist concentrations, while the EC50 remains largely unaffected. Our results, summarized in Figure 7, show a significant depression of the upper plateaus of the UTP curves with increasing concentrations of 61. The reduction in the maximum receptor activation is successively more pronounced with rising concentrations of 61, and a complete blockade of UTP-mediated receptor activation is observed at a concentration of 15 μM of 61. In the calcium mobilization assay, the cells were first incubated with the antagonist 61 for 30 min. The agonist UTP was subsequently injected into the cell suspension, and the assay signal was read immediately thereafter. Because there is no simultaneous incubation with both agonist and antagonist and the measurement of the signal is very fast, the assay conditions are likely to 3027



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Analysis of the Human P2Y4 Receptor Model. Two P2Y receptor subtypes, the more distantly related P2Y12 and the more closely related P2Y1 receptors, had been crystallized and X-ray structures were obtained in complex with agonists (P2Y12) and antagonists (P2Y1, P2Y12).54−56 On the basis of these structures, the molecular mechanism of activation of the human P2Y1 receptor was recently analyzed by molecular dynamics simulations, which explained the essential role of the amino acids Asp204 in extracellular loop 2 (ECL2) and Arg310 in the transmembrane region 7 (TM VII).57 The two amino acids Asp204 and Arg310 form an ionic lock and are stabilized in the inactive state by P2Y1 antagonists. The ionic lock is broken during the activation of the receptor by the agonist adenosine diphosphate (ADP). We constructed a homology model of the P2Y4 receptor based on the P2Y1 receptor. In our inactive state homology model of the human P2Y4 receptor, an ionic lock between Asp187 and Arg292 was formed, suggesting a similar mechanism of activation as in the human P2Y1 receptor. Two ligand binding sites located toward the extracellular regions of the P2Y4 receptor were identified in the homology model (Figure 8). The binding site denoted as “A” with a volume of 190 Å3 was observed upon the downward movement of the ECL2. This binding site “A” is comparable to the binding site of the noncompetitive antagonist 2-iodo-N6-methyl-(N)methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2500, 72) in the human P2Y1 receptor with a size of 214 Å3. The second binding site in the homology model of the P2Y4 receptor, denoted as “O”, is formed in the region comparable to the orthosteric binding site identified in the crystal structure of the human P2Y12 receptor in complex with the agonist 2methylthioadenosine-5′-diphosphate (2MeSADP, 71) and in the molecular dynamics simulation study of the human P2Y1

Figure 7. Concentration−response curves of UTP on the P2Y4 receptor after preincubation with fixed concentrations of 61. The results were obtained using the calcium mobilization assay with 1321N1 astrocytoma cells recombinantly expressing the human P2Y4 receptor. EC50 values are not significantly different from each other (p > 0.05, unpaired t-test). The EC50 values and maximum receptor activation are shown in the Supporting Information.

be under nonequilibrium conditions. Nevertheless, our data indicate that 61 may achieve P2Y4 receptor antagonism via a noncompetitive mechanism, but competitive antagonism cannot be completely ruled out at present. Interestingly, on the P2Y12 receptor, we previously found that anthraquinone derivatives of this series act as competitive antagonists.30 However, the percentage of sequence shared by the P2Y4 and P2Y12 receptors is only 25% and, consequently, significant differences exist between these two receptors (also with respect to orthosteric ligand preferences and G protein coupling). To get more insight into the interaction of the anthraquinone derivatives with the P2Y4 receptor on a molecular level, we decided to perform docking studies.

Figure 8. (a) Crystal structure of the human P2Y12 receptor (light red) in complex with the agonist 2-methylthio-ADP (2MeSADP, 71). (b) Homology model of the human P2Y4 receptor (light green) with the schematic representation of the orthosteric binding site (denoted “O”) and the allosteric binding site (denoted “A”). (c) Crystal structure of the human P2Y1 receptor (light blue) in complex with the noncompetitive antagonist 2iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2500, 72). The receptors are represented as cartoon models, and the small molecules 71 and 72 are represented as space fill models. 3028



Article

Figure 9. Proposed binding modes of Reactive Blue 2 (RB-2) in our homology model of the P2Y4 receptor. Docked poses of RB-2 (a) parasubstituted and (b) meta-substituted with the important residues in the putative binding pocket of the human P2Y4 receptor and their 2D interaction diagrams shown in (c) and (d). The human P2Y4 receptor model (dark cyan) is depicted in cartoon representation, and the amino acid residues (white) and RB-2 (marine blue) are presented as stick models. The oxygen atoms are colored in red, nitrogen atoms in blue and sulfur atoms in orange. None of the interacting residues are highly conserved for the subtypes analyzed in selectivity studies (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12; see Supporting Information, Figure 61).

Ring B may be stabilized by π−π-stacking with Phe29 from the N-terminal region of the receptor. The sulfonate group in position 2 of ring C likely binds to Lys179 and Arg272, forming strong electrostatic and hydrogen bonding interactions. The carbonyl groups at positions 9 and 10 of the anthraquinone core, the amino group at position 1, and the amine linker between rings C and D were not observed to form any specific interactions with the amino acids of the human P2Y4 receptor. However, the amino group at position 1 of the anthraquinone ring system probably forms intramolecular H-bond interactions with the carbonyl group at position 9. The sulfonate group at ring D forms strong electrostatic interactions with Lys34, Lys289, and Arg292, and possibly with Asp187, according to our model. Ring E was found to occupy the binding pocket formed by His186 and Thr188 of ECL2, Tyr268 and Arg272 of TM VI, and Tyr288 of TM VII. Ring F is bound by the aromatic residues Tyr116, Tyr197, and Tyr269 between TM

receptor with the agonist ADP.56,57 In our inactive state model of the human P2Y4 receptor, the binding site “O” is limited in size due to the downward movement of the ECL2. However, after its upward movement, it could accommodate the endogenous agonists UTP and ATP, respectively. The experimental data for the P2Y4 receptor antagonists developed in this study suggested a noncompetitive mode of inhibition. Hence, we selected binding site “A” for compound docking to probe whether 61 and analogues may display a similar mode of receptor inactivation as MRS2500 (72) in the P2Y1 receptor. Docking Studies of Reactive Blue 2 (RB-2). The putative binding modes of the para- and meta-sulfo-substituted isomers (ring F) of RB-2 are shown in Figure 9. The binding poses showed that the two derivatives have the same orientation in the binding pocket “A” formed between ECL2 and TM regions V, VI, and VII. Ring A of RB-2 appears to be completely exposed to the extracellular space of the human P2Y4 receptor. 3029



Article

Figure 10. Proposed binding mode of 61 in our homology model of the P2Y4 receptor. (a) Docked pose of 61 with the important residues in the putative binding pocket of the human P2Y4 receptor. (b) The putative binding pocket is shown in a surface model; the 2D interaction diagram is shown in (c). The human P2Y4 receptor model (dark cyan) is displayed in cartoon representation, and the amino acid residues (white) and compound 61 (marine blue) are shown as stick models. The oxygen atoms are colored in red, nitrogen atoms in blue, and sulfur atoms in orange. As for RB-2, none of the residues interacting with 61 are highly conserved for the subtypes analyzed in selectivity studies (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12; see Supporting Information, Figure 61).

regions V and VI. The sulfonate group on ring F (para- or meta-substituted) interacts with Arg265 through electrostatic interactions and additionally stabilizes the ring in the binding pocket through cation−π interactions as observed in the docking study. Docking Studies of the New, Potent P2Y4 Receptor Antagonists. To propose binding modes for the anthraquinone derivatives that were optimized for P2Y4 receptor blockade, and to rationalize the determined potency values, we selected compounds 7, 42, and 61−64 for performing molecular docking studies on the homology model of the human P2Y4 receptor. The putative binding pose of the most potent antagonist 61 in the binding pocket and its 2D interaction diagram are shown in Figure 10. Similar to RB-2,

compound 61 can be well accommodated in binding pocket “A”. Ring A of 61 faces toward the extracellular space and rings B and C form hydrophobic interactions with Phe29 and Leu184. The sulfonate group in ring C could make strong electrostatic interactions with Lys34, Asp187, and Arg292 present in the pocket formed by Lys34, Tyr94, Asp187, Lys289, and Arg292 of the ECL2 and TMs I, VI, and VII. Through its strong electrostatic interactions, the sulfonate group may act as an anchor for the compound and stabilize it in a vertical position through hydrophobic interactions of ring B and C with Phe29 and Leu184. The carbonyl group at position 9 of the anthraquinone ring system likely interacts with Asn99 through hydrogen bonding, and the carbonyl group at position 10 and the amino group at position 1 may form weak hydrogen bond 3030



Article

Figure 11. Comparative overlay of the docked poses of para-substituted RB-2 (in blue) and compound 61 (in red) in the binding pocket of the human P2Y4 receptor model. The overlap of the ring system of RB-2 and 61 is shown in the schematic representation on the top.

increase the hydrophobic interactions with the aromatic residues and promote the tight binding of the compound inside the binding pocket of the P2Y4 receptor. Among different substituents, 2,4-dimethylphenylthio-substitution (61, 0.233 μM) showed slightly higher potency as compared to 2,5and 3,4-dimethylphenylthio-substituted derivatives (62, 0.395 μM; 63, 0.482 μM). The putative binding poses and 2D interaction diagrams of 62 and 63 are shown in Supporting Information, Figure 56. In case of 64, tight binding was achieved with an extended linker. Additionally, Arg265 is located at the bottom of the pocket occupied by ring E and might interact with the pyridine group of 64. The putative binding pose and 2D interaction diagram of 64 are shown in Supporting Information, Figure 57. As a next step, we compared the binding poses of RB-2 and compound 61 (see overlay of RB-2 and 61 in Figure 11). Ring B of RB-2 and ring A of 61 form hydrophobic aromatic interactions with Phe29. The sulfonate groups in ring D of RB2 correlates with the sulfonate group in ring C of 61; they are believed to interact with Lys34 and Arg292. Ring E of RB-2 and ring D of 61 occupy the same binding pocket formed by the amino acid residues His186, Thr188, Tyr268, Arg272, and Tyr288. A comparison of ring F of RB-2 with ring E of 61 shows that the sulfonate group in ring F probably displays electrostatic interactions with Arg265 in the bottom part of the hydrophobic pocket. The dimethyl substitution of 61 may lead to a tightening of the binding pocket through hydrophobic interactions with the aromatic residues instead. Thus, our homology model explains the positive contributions of lipophilic terminal moieties on the anthraquinone derivatives as well as those of a polar sulfonate moiety as in RB-2. A further important observation regarding the binding poses of the antagonists in the binding pocket of the human P2Y4 receptor relate to the orientation and interactions of Asp187 and Arg292, which correspond to Asp204 and Arg310 in the human P2Y1 receptor. This supports the hypothesis that the mode of P2Y4 receptor inhibition by anthraquinone derivatives is similar to that described for 72 at the P2Y1 receptor. They

interactions with Arg272 and Lys34, respectively. Additionally, the amino group at position 1 of the anthraquinone probably forms intramolecular interactions with the carbonyl group at position 9 and the sulfonate oxygen at ring C. The importance of the amino group at position 1 was confirmed for compounds 68−70 by replacing the amino group with a hydrogen atom. This led to a significant drop in inhibitory potency. The amine linker between ring C and D may form weak hydrogen bond interactions with His186. We hypothesize that the sulfonate group at ring C forms strong electrostatic interactions with Arg292; it is essential for high potency of the anthraquinone derivatives. The presence of an acidic (carboxylic) group at ring D decreased potency (see 30−32, 46, 67, and 70). A possible explanation is that both acidic structures compete for the same binding subpocket interacting with Arg292, which might lead to different binding modes. Furthermore, the binding pocket of ring D appears to be limited in space with the subpocket formed by His186, Thr188 of ECL2, and Tyr268, Arg272 and Tyr288 of TMs VI and VII. In this pocket, phenyl substitution is well tolerated, as opposed bulky groups, for example, the naphthly group (compounds 8−10). This was additionally supported by the linker preferences between rings D and E: S > O > NH > CH2. Thiophenoxy derivatives were most potent, likely because the orientation provided by a sulfur linker is required for positioning ring E into the hydrophobic pocket. Except for compound 62, ring D of the docked compounds showed the same orientation within the subpocket (Supporting Information, Figure 54). The terminal ring E present in 61−63 and 64 was found to occupy the pocket formed by Tyr116 of TM III, Phe194, Tyr197, and Val198 of TM V, and Arg265, Tyr268, and Tyr269 of TM IV. Because of a large number of aromatic residues, compounds with a ring E may be tightly packed within the pocket, resulting in somewhat improved potency in comparison to the compounds without ring E. The putative binding modes of compounds 7 and 42 without ring E, and their 2D interaction diagrams are shown in Supporting Information, Figure 55. The dimethyl substitutions at ring E are thought to 3031



Article

53, 58−64, and 67−69) were newly prepared in analogy to described methods27−29,39 with modifications as described below. Purification of Reactive Blue 2 (RB-2). RB-2 (200 mg) was dissolved in 10 mL of deionized water and injected into a flash column chromatography type Sepacore glass column C-690 (ID 26 mm × 500 mm) two-thirds filled with reversed phase-18 silica gel (40−63 μm, Merck) using water as eluent. The polarity was gradually decreased by increasing the concentration of methanol (5, 10, 20, 40, and 60%). The pooled blue-colored fractions were collected and evaporated under vacuum to remove the methanol, and the remaining water was subsequently removed by lyophilization to yield 108 mg (54%) of RB2 P (2′) as a blue powder. The chemical structure and purity were confirmed by RP-TLC, LC-MS (>96%), and NMR. General Procedure A: Preparation of 4-Substituted 1-Aminoanthraquinone-2-sulfonate Derivatives (14, 19−21, 24, 25, 33−35, 37, 39−44, 49, 52, 53, 58−64, and 67). To a 5 mL microwave reaction vial, equipped with a magnetic stirring bar, were added 1amino-4-bromo substituted anthraquinone compounds (bromaminic acid sodium salt 6a, or 1-amino-4-bromo-2-methylanthraquinone 6b) (0.1−0.3 mmol) and the appropriate aniline or amine derivative (1.5− 9.0 equiv), followed by a buffer solution of Na2HPO4 (pH 9.6) (4.5 mL) and NaH2PO4 (pH 4.2) (0.5 mL) and a finely powdered elemental copper (0.002−0.003 g, 5−10 mol %). The mixture was capped and irradiated in the microwave oven (80−100 W) for 5−24 min at 100−120 °C. The reaction mixture was cooled down to room temperature (rt), and the product was purified using the following procedure. The contents of the vial were filtered to remove the elemental copper. Then ca. 200 mL of water was added to the filtrate and the aqueous solution was extracted with dichloromethane (200 mL). The extraction procedure was repeated until the dichloromethane layer became colorless (2−3 times). The aqueous layer was reduced by rotary evaporation to a volume of 10−20 mL, which was subsequently submitted to flash column chromatography using RP-18 silica gel and water as an eluent. The polarity of the eluent was then gradually decreased by the addition of acetone in the following steps: 5, 10, 20, 40, and 60%. Fractions containing blue product were collected. For some compounds the last step of purification (RP-18 flash chromatography) had to be repeated two to three times to obtain pure product (≥95% purity as determined by LC-MS, Table 1). The pooled product-containing fractions were evaporated under vacuum to remove the acetone and reduce water volume. The remaining water was subsequently removed by lyophilization to yield (up to 80%) of the product as blue powder (Scheme 1 and Table 1). General Procedure B: Preparation of 4-Substituted Anthraquinone-2-sulfonate Derivatives (68 and 69). To a 50 mL roundbottom flask equipped with a magnetic stirring bar, 0.1 mmol of 1aminoanthraquinone derivative (12 or 27) were added, followed by 5 mL of 1 molar hydrochloric acid. The solution was cooled to 0−5 °C in an ice bath, and a previously cooled solution of NaNO2 (13.8 mg, 0.2 mmol, 2 equiv) in 0.5 mL of distilled water was added dropwise. After 5 min, the mixture was allowed to warm up to rt followed by addition of 30 mg of zinc powder (1.0 mmol, 10 equiv) and 5 mL of ethanol. The resulting mixture was then allowed to stir at rt for ca. 30 s. The mixture was filtered off, and the purple-colored filtrate was then purified by flash column chromatography on a reversed phase silica gel (RP-18) using a gradient of acetone in water (5% and 20%) as the eluent. Fractions containing the purple product were collected and evaporated in vacuum to remove acetone and decrease the volume of water to ca. 20 mL. Complete drying was achieved with a freeze-dryer, affording purple-colored products 68 and 69 in 97% and 38 yield, respectively (Scheme 3 and Table 1). Sodium 1-Amino-4-(2-chlorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (14). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 2chloroaniline (51 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (36% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.20 (m, 1H, 4′-H), 7.42 (m, 2H, 5′-H, 6′-H), 7.61 (dd, 1H, 3′-H), 7.86 (m, 2H, 6-H, 7-H), 7.91 (s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.00 (br, 2H, 1-NH2), 11.95 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 109.61, 112.91, 122.79, 123.81,

stabilize the ionic lock and prevent the movement of TMs VI and VII required for receptor activation. Our docking results are consistent with the experimental data and the SARs of anthraquinone derivatives. The homology model of the human P2Y4 receptor will be useful for future compound optimization and allow rational ligand design and virtual screening approaches.

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CONCLUSIONS In conclusion, we have synthesized a library of RB-2-related anthraquinone derivatives and successfully optimized them as selective antagonists of the human P2Y4 receptor. To the best of our knowledge, compound 61 (sodium 1-amino-4-[4-(2,4dimethylphenylthio)-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most potent P2Y4 receptor antagonist known to date, with an IC50 value of 233 nM. Our results suggest that 61 exerts its antagonistic effect on the P2Y4 receptor through a noncompetitive mechanism. This was rationalized by the docking of 61 to a homology model of the human P2Y4 receptor based on the recently published X-ray structure of the P2Y1 receptor in complex with allosteric antagonists. As a continuation of our previous work on anthraquinone derivatives, the current results confirm the validity of RB-2 as a privileged structure in the field of purinergic signaling. Furthermore, by fine-tuning the structure, it is possible to develop subtype-specific ligands as valuable pharmacological tool compounds.

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EXPERIMENTAL SECTION

Chemistry. Material and Methods. All materials were used as purchased (Acros, Alfa Aesar, Merck, or Sigma-Aldrich, Germany). 3Propylaniline was prepared according to the method of Rasmussen et al.58 Thin-layer chromatography was performed using TLC aluminum sheets silica gel 60 F254 or TLC aluminum sheets reversed phase (RP) silica gel 18 F254 (Merck, Darmstadt, Germany). Colored compounds were visible at daylight; other compounds were visualized under UV light (254 nm). Flash chromatography was performed on a Büchi system using silica gel RP-18 (Merck, Darmstadt, Germany). 1H and 13 C NMR data were collected on either a Bruker Avance 500 MHz NMR spectrometer at 500 MHz (1H) or 126 MHz (13C), respectively, or a 600 MHz NMR spectrometer at 600 MHz (1H) or 151 MHz (13C), respectively. DMSO-d6 was used as a solvent. Chemical shifts are reported in parts per million (ppm) relative to the deuterated solvent, i.e., DMSO, δ 1H 2.49 ppm; 13C 39.7 ppm, coupling constants J are given in hertz, and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), sext (sextet), m (multiplet), and br (broad). The purities of isolated products were determined by ESI−mass spectra obtained on an LC-MS instrument (Applied Biosystems API 2000 LC-MS/MS, HPLC Agilent 1100) using the following procedure: the compounds were dissolved at a concentration of 0.5 mg/mL in H2O:MeOH = 1:1, containing 2 mM NH4CH3COO. Then, 10 μL of the sample was injected into an HPLC column (Phenomenex Luna 3 μ C18, 50 mm × 2.00 mm). Elution was performed with a gradient of water:methanol (containing 2 mM NH4CH3COO) from 90:10 to 0:100 starting the gradient immediately at a flow rate of 250 μL/min for 15 min, followed by washing with 100% methanol for another 15 min. UV absorption was detected from 200 to 950 nm using a diode array detector. The purity of the compounds proved to be ≥95%. For microwave reactions, a CEM Focused Microwave Synthesis Type Discover apparatus was used. A freeze-dryer (CHRIST ALPHA 1-4 LSC) was used for lyophilization. The synthesis and analysis of compounds 7−12, 13, 15−18, 22, 23, 26−32, 36, 38, 45− 48, 50, 51, 65−66, and 70 were previously described.24,27−29,33,36,38−40 All other compounds (14, 19−21, 24, 25, 33−35, 37, 39−44, 49, 52, 3032



Article

125.48, 126.24, 126.50, 128.30, 130.48, 133.06, 133.56, 133.64, 134.32, 136.77, 139.32, 142.66, 144.70, 182.20, 183.47. LC-MS (m/z): 446 [M − Na+ + NH4+]+, 429 [M − Na+ + H+]+, 427 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(2-methylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (19). Reaction conditions according to general procedures A: Compound 6a (81 mg, 0.2 mmol), 2methylaniline (43 mg, 0.4 mmol). MW conditions: 10 min, 120 °C, 100 W; pressure up to 10 bar. Yield 80%. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(3-methylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (20). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 3methylaniline (43 mg, 0.4 mmol). MW conditions: 10 min, 120 °C, 100 W; pressure up to 10 bar. Yield 70%. Purity by HPLC-UV (254 nm) ESI-MS: 99.1%. Sodium 1-Amino-4-(4-methylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (21). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4methylaniline (64.3 mg, 0.6 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Yield 72%. Purity by HPLC-UV (254 nm) ESI-MS: 100%. Sodium 1-Amino-4-(4-ethylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (24). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 4-ethylaniline (48.5 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (64% yield), mp >300 °C. 1 H NMR (500 MHz): δ 1.22 (t, 3H, 4′-CH2CH3), 2.64 (q, 2H, 4′CH2CH3), 7.20 (d, 2H, 2′-H, 6′-H), 7.29 (d, 2H, 3′-H, 5′-H), 7.84 (m, 2H, 6-H, 7-H), 7.98 (s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.10 (br, 2H, 1-NH2), 12.07 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 15.64, 27.75, 109.18, 111.03, 122.73, 123.61, 126.05, 126.15, 129.12, 132.85, 133.18, 133.76, 134.28, 136.81, 140.43, 141.59, 143.04, 144.39, 181.84, 182.30. LC-MS (m/z): 440 [M − Na+ + NH4+]+, 423 [M − Na+ + H+]+, 421 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(3-propylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (25). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 3propylaniline (61 mg, 0.45 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (35% yield), mp >300 °C. 1H NMR (500 MHz): δ 0.91 (t, 3H, −CH2CH2CH3), 1.63 (sext, 2H, −CH2CH2CH3), 2.58 (t, 2H, −CH2CH2CH3), 7.03 (d, 1H, 6′-H), 7.10 (m, 2H, 2′-H, 4′-H), 7.35 (t, 1H, 5′-H), 7.85 (m, 2H, 6-H, 7-H), 8.05 (s, 1H, 3-H), 8.28 (m, 2H, 5-H, 8-H), 12.07 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 13.65, 23.91, 37.91, 109.06, 111.19, 120.32, 122.79, 123.02, 124.59, 125.90, 126.00, 129.41, 132.71, 133.09, 133.57, 134.12, 139.05, 140.93, 142.79, 143.94, 144.30, 181.72, 182.32. LC-MS (m/z): 454 [M − Na+ + NH4+]+, 437 [M − Na+ + H+]+, 435 [M − Na+ + H+]−. Purity by HPLC-UV (220−700 nm) ESI-MS: 97.7%. Sodium 1-Amino-4-(2,3-dimethylphenylamino)-9,10-dioxo-9,10dihydroanthracene-2-sulfonate (33). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 2,3dimethylaniline (48.5 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Yield 49%. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(2,4-dimethylphenylamino)-9,10-dioxo-9,10dihydroanthracene-2-sulfonate (34). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 2,4dimethylaniline (48.5 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Yield 60%. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(2,5-dimethylphenylamino)-9,10-dioxo-9,10dihydroanthracene-2-sulfonate (35). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 2,5dimethylaniline (48.5 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Yield 50%. Purity by HPLC-UV (254 nm) ESI-MS: 99.5%. Sodium 1-Amino-4-(3,4-dimethoxyphenylamino)-9,10-dioxo9,10-dihydroanthracene-2-sulfonate (37). Reaction conditions ac-

cording to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 3,4-dimethoxyaniline (92 mg, 0.6 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (46% yield), mp >300 °C. 1H NMR (500 MHz): δ 3.78 (s, 3H, −OCH3), 3.80 (s, 3H, −OCH3), 6.83 (dd, 1H, 6′-H, J2′,6′ 2.45 Hz, J5′,6′ 8.5 Hz), 6.94 (d, 1H, 2′-H, J2′,6′ 2.45 Hz), 7.03 (d, 1H, 5′-H, J5′,6′ 8.5 Hz), 7.85 (m, 2H, 6-H, 7-H), 7.98 (s, 1H, 3-H), 8.28 (m, 1H, 5-H, 8-H), 12.08 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 55.60, 55.74, 108.88, 110.34 (C-4a), 112.55, 115.94, 122.79, 125.82, 125.97, 131.96, 132.63, 132.90, 133.65, 134.12, 142.21, 142.91, 144.14, 146.42, 149.49, 181.57, 181.80. LCMS (m/z): 472 [M − Na+ + NH4+]+, 455 [M − Na+ + H+]+, 453 [M − Na+ + H+]−. Purity by HPLC-UV (220−700 nm) ESI-MS: 98.5%. Sodium 1-Amino-4-(3-methoxy-4-methylphenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (39). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 3-methoxy-4-methylaniline (82.3 mg, 0.6 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (44% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.17 (s, 1H, −CH3), 3.81 (s, 3H, −OCH3), 6.77 (dd, 1H, 6′-H, J2′,6′ 2.0 Hz, J5′,6′ 7.9 Hz), 6.89 (d, 1H, 2′-H, J2′,6′ 2.0 Hz), 7.19 (d, 1H, 5′H, J5′,6′ 7.9 Hz), 7.85 (m, 2H, 6-H, 7-H), 8.10 (s, 1H, 3-H), 8.28 (m, 1H, 5-H, 8-H), 12.10 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 15.69, 55.49, 106.40, 109.16, 111.05, 115.03, 122.20, 123.15, 126.03, 126.14, 131.08, 132.84, 133.18, 133.75, 134.28, 138.15, 141.46, 142.93, 144.40, 158.21, 181.83, 182.28. LCMS (m/z): 456 [M − Na+ + NH4+]+, 439 [M − Na+ + H+]+,. 437 [M − Na+ + H+]− Purity by HPLC-UV (220− 700 nm) ESI-MS: 98.3%. Sodium 1-Amino-4-(4-chloro-2-methylamino)-9,10-dioxo-9,10dihydroanthracene-2-sulfonate (40). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 4-chloro-2methylaniline (56.6 mg, 0.4 mmol). MW conditions: 24 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (36% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.28 (s, 3H, 2′-CH3), 7.27 (d, 1H, 6′-H, J5′,6′ 8.5 Hz), 7.33 (dd, 1H, 5′-H, J3′,5′ 2.4 Hz, J5′,6′ 8.5 Hz), 7.46 (d, 1H, 3′-H, J3′,5′ 2.4 Hz), 7.70 (s, 1H, 3-H), 7.84 (m, 2H, 6-H, 7-H), 8.26 (m, 2H, 5-H, 8-H), 10.05 (br, 2H, 1-NH2), 11.88 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 17.69, 109.31, 111.46, 122.53, 125.89, 126.11, 126.20, 126.91, 129.03, 130.85, 132.92, 133.36, 133.67, 134.31, 134.70, 136.97, 131.40, 143.01, 144.36, 181.95, 182.83. LC-MS (m/z): 460 [M − Na+ + NH4+]−, 443 [M − Na+ + H+]+, 441 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(4-chloro-3-methylphenylamino)-9,10-dioxo9,10-dihydroanthracene-2-sulfonate (41). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 4chloro-3-methylaniline (56.6 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (40% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.34 (s, 3H, CH3), 7.13 (dd, 1H, 6′-H, J2′,6′ 2.7 Hz, J5′,6′ 8.5 Hz), 7.27 (d, 1H, 2′-H, J2′,6′ 2.7 Hz), 7.44 (d, 1H, 5′-H, J5′,6′ 8.5 Hz),7.84 (m, 2H, 6-H, 7-H), 7.96 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-H), (br, 2H, 1-NH2 not detectable), 11.90 (br, 1H, 4-NH). 13C NMR (126 MHz): δ 19.80, 109.38, 111.96, 122.16, 122.86, 125.82, 126.09, 126.18, 128.69, 129.94, 132.96, 133.40, 133.63, 134.27, 137.02, 138.47, 140.48, 142.84, 144.52, 182.02, 182.82. LC-MS (m/z): 460 [M − Na+ + NH4+], 443 [M − Na+ + H+]+, 441 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 99%. Sodium 1-Amino-4-(4-hydroxy-3-methylphenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (42). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-amino-2-methylphenol (111 mg, 0.9 mmol). MW conditions: 20 min, 100 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (40% yield), mp >300 °C. 1H.NMR (500 MHz): δ 2.15 (s, 3H, CH3), 6.86 (d, 1H, 5′-H, J5′,6′ 8.4 Hz), 6.93 (dd, 1H, 6′-H, J5′,6′ 8.4, J2′,6′ 2.6 Hz), 7.01 (d, 1H, 2′-H, J2′,6′ 2.6 Hz), 7.81 (s, 1H, 3H), 7.84 (m, 2H, 6-H, 7-H), 8.28 (m, 2H, 5-H, 8-H).13C NMR (126 MHz): δ 15.94, 108.73, 109.68, 115.32, 122.57, 123.39, 125.23, 125.79, 125.94, 127.37, 129.58, 132.54, 132.73, 133.74, 134.11, 143.07, 143.28,143.97, 153.42, 181.41, 181.46. LC-MS (m/z): 425 [M − Na+ + H+]+, 423 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 96%. 3033



Article

Sodium 1-Amino-4-[4-(4-fluorophenoxy)phenylamino]-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (58). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-(4-fluorophenoxy)aniline (131.5 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (41% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.06 (d, 2H, 3′-H, 5′-H), 7.13 (dd, 2H, 2″-H, 6″-H), 7.24 (dd, 2H, 3″H, 5″-H), 7.30 (d, 2H, 2′-H, 6′-H), 7.84 (m, 2H, 6-H, 7-H), 7.94 (s,1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.11 (br, 2H, 1-NH2), 12.03 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 109.2, 111.1, 116.6, 116.8, 119.4, 120.8, 120.9, 122.6, 125.5, 126.0, 126.1, 132.9, 133.2, 133.7, 134.0, 134.6, 141.6, 143.1, 144.4, 152.7, 154.3, 157.4, 159.4, 181.9, 182.4. LCMS (m/z): 522 [M − Na+ + NH4+]+, 505 [M − Na+ + H+]+, 503 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 99.5%. Sodium 1-Amino-4-[4-(4-chlorophenoxy)phenylamino]-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (59). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-(4-chlorophenoxy)aniline (141.4 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (25% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.10 (m, 4H, 3′-H, 5′-H, 2″-H, 6″-H), 7.32 (dd, 2H, 3″-H, 5″-H), 7.44 (d, 2H, 2′-H, 6′-H), 7.85 (m, 2H, 6-H, 7-H), 7.96 (s,1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.11 (br, 2H, 1-NH2), 12.03 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 109.3, 111.3, 120.2, 120.4, 122.6, 125.4, 126.1, 126.2, 127.4, 130.1, 132.9, 133.3, 133.7, 134.3, 135.2, 141.4, 143.1, 144.4, 153.2, 155.9, 181.9, 182.5. LC-MS (m/z): 519 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 98.6%. Sodium 1-Amino-4-[4-(4-bromophenoxy)phenylamino]-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (60). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-(4-bromophenoxy)aniline (190 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (17% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.03 (d, 2H, 3′-H, 5′-H) 7.12 (d, 2H, 2″-H, 6″-H), 7.33 (dd, 2H, 3″-H, 5″-H), 7.56 (d, 2H, 2′-H, 6′-H), 7.85 (m, 2H, 6-H, 7-H), 7.96 (s,1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.10 (br, 2H, 1-NH2), 12.03 (s, 1H, 4NH). 13C NMR (126 MHz): δ 109.3, 111.3, 115.2, 120.3, 120.7, 122.6, 125.3, 126.1, 126.2, 132.9, 133.0, 133.3, 135.3, 141.4, 144.4, 153.1, 156.5, 181.9, 182.5. LC-MS (m/z): 565 [M − Na+ + H+]+, 563 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 96.1%. Sodium 1-Amino-4-[4-(2,4-dimethylphenylthio)phenylamino]9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (61). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-[(2,4-dimethylphenyl)thio]aniline (137.6 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (10% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.31, 2.28 (2s, 6H, 2CH3), 7.04, 7.20 (m, 7H, 2′-H, 3′H, 5′-H, 6′-H, 3″-H, 5″-H, 6″-H), 7.84 (m, 2H, 6-H, 7-H), 7.98 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-H), 10.05 (br, 2H, 1-NH2), 11.95 (s, 1H, 4-NH). 13C NMR: δ 20.2, 20.7, 109.4, 112.0, 122.9, 123.7, 126.1, 126.2, 127.9, 129.5, 130.4, 130.9, 131.7, 132.9, 133.3, 133.6, 134.2, 138.1, 138.2, 139.5, 140.2, 142.8, 144.5, 182.0, 182.7. LC-MS (m/z): 531 [M − Na+ + H+]+, 529 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 96.1%. Sodium 1-Amino-4-[4-(2,5-dimethylphenylthio)phenylamino]9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (62). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-[(2,5-dimethylphenyl)thio]aniline (137.6 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (7% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.23, 2.30 (2s, 6H, 2CH3), 7.08, 7.23 (m, 7H, 2′-H, 3′H, 5′-H, 6′-H, 3″-H, 4″-H, 6″-H), 7.84 (m, 2H, 6-H, 7-H), 8.00 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-H), 10.07 (br, 2H, 1-NH2), 11.94 (s, 1H, 4-NH). 13C NMR: δ 19.8, 20.6, 109.4, 112.2, 123.0, 123.7, 126.1, 126.2, 128.9, 129.9, 130.7, 131.4, 132.6, 133.0, 133.2, 133.4, 133.6, 134.3, 135.9, 136.4, 138.6, 140.1, 142.8, 144.6, 182.0, 182.8. LC-MS (m/z): 531 [M − Na+ + H+]+, 529 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 98.0%. Sodium 1-Amino-4-[4-(3,4-dimethylphenylthio)phenylamino]9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63). Reaction con-

Sodium 1-Amino-4-(4-fluoro-3-methoxyphenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (43). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-fluoro-3-methoxylaniline (84.7 mg, 0.6 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (43% yield), mp >300 °C. 1H NMR (500 MHz): δ 3.86 (s, 3H, −OCH3), 6.85 (m, 1H, 6′-H), 7.14 (dd, 1H, 2′-H, 4JH−F 7.8 Hz, 4J2′,6′ 2.5 Hz), 7.27 (dd, 1H, 5′-H, 3JH−F 11.35 Hz, 4J5′,6′ 8.6 Hz), 7.86 (m, 2H, 6-H, 7-H), 8.00 (s, 1H, 3-H), 8.28 (m, 1H, 5-H, 8H), 11.96 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 56.52, 109.07, 109.79, 111.22, 115.36, 116.37, 122.72, 125.88, 126.01, 132.74, 133.14, 133.52, 134.12, 135.89, 141.06, 142.77, 144.26, 147.78, 149.65, 181.77, 182.40. LCMS (m/z): 460 [M − Na+ + NH4+]+, 443 [M − Na+ + H+]+, 441 [M − Na+ + H+]−. Purity by HPLC-UV (220−700 nm) ESI-MS: 99.3%. Sodium 1-Amino-4-(4-chloro-3-methoxyphenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (44). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-chloro-3-methoxylaniline (94.6 mg, 0.6 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (40% yield), mp >300 °C. 1H NMR (500 MHz): δ 3.88 (s, 3H, −OCH3), 6.87 (dd, 1H, 6′-H, J2′,6′ 2.4 Hz, J5′,6′ 8.45 Hz), 7.10 (d, 1H, 2′-H, J2′,6′ 2.4 Hz), 7.44 (d, 1H, 5′-H, J5′,6′ 8.45 Hz), 7.86 (m, 2H, 6-H, 7-H), 8.10 (s, 1H, 3-H), 8.27 (m, 1H, 5-H, 8-H), 11.90 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 56.14, 107.60, 109.26, 112.08, 115.13, 116.00, 123.08, 125.92, 126.03, 130.31, 132.81, 133.28, 133.43, 134.10, 139.67, 139.80, 142.51, 144.43, 155.23, 181.88, 182.74. LC-MS (m/z): 476 [M − Na+ + NH4+]+, 459 [M − Na+ + H+]+, 457 [M − Na+ + H+]−. Purity by HPLC-UV (220−700 nm) ESI-MS: 99.7%. Sodium 1-Amino-4 (2-carboxy-3-fluorophenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (49). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 2-amino-6-fluorobenzoic acid (93 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (40% yield), mp >300 °C. 1H NMR (500 MHz): δ 6.78 (dd, 1H, 5′-H), 6.94 (d, 1H, 6′-H), 7.15 (dd, 1H, 4′-H), 7.82 (m, 2H, 6-H, 7-H), 8.10 (s,1H, 3-H)), 8.25 (m, 2H, 5-H, 8-H), 10.05 (br, 2H, 1-NH2), 12.07 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 109.67, 110.09, 113.18, 116.83, 124.46, 126.27, 126.04, 127.18, 127.22, 132.82, 133.13, 133.86, 134.22, 138.59, 139.03, 141.96, 144.59, 159.54, 166.02, 181.9, 182.2. (m/z): 474 [M − Na+ + NH4+]+, 457 [M − Na+ + H+]+, 455 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 98%. Sodium 1-Amino-4-(2-carboxy-4-hydroxyphenylamino)-9,10dioxo-9,10-dihydroanthracene-2-sulfonate (52). Reaction conditions according to general procedure A: Compound 6a (81 mg, 0.2 mmol), 2-amino-5-hydroxybenzoic acid (61.3 mg, 0.4 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder, (68% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.00 (dd, 1H, 5′-H, J3′,5′ 2.9 Hz, J5′,6′ 8.7 Hz), 7.17 (d, 1H, 6′-H, J5′,6′ 8.7 Hz), 7.33 (d, 1H, 3′-H, J3′,5′ 2.9 Hz), 7.83 (m, 2H, 6-H, 7-H), 7.90 (s, 1H, 3-H), 8.24 (m, 2H, 5-H, 8-H), 9.7 (br, 2H, 1-NH2), 12.27 (s, 1H, 4-NH). 13C NMR (126 MHz): δ 109.54, 112.69, 117.20, 120.36, 123.80, 124.19, 125.33, 126.03, 126.11, 132.04, 132.84, 133.09, 133.90, 134.24, 140.03, 142.17, 144.44, 153.4, 167.33, 181.88, 182.06. LC-MS (m/z): 555 [M − Na+ + H+]+, 553 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm)-ESI-MS: 100%. Sodium 1-Amino-4-(2-carboxy-4-nitrophenylamino)-9,10-dioxo9,10-dihydroanthracene-2-sulfonate (53). Reaction conditions according to general procedure A: Compound 6a (60.6 mg, 0.15 mmol), 2-amino-5-nitrobenzoic acid (246 mg, 1.35 mmol). MW conditions: 20 min, 110 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (21% yield), mp >300 °C. 1H NMR (500 MHz): δ 7.10 (d, 1H, 6′-H, J5′,6′ 9.2 Hz) 7.86 (m, 2H, 6-H and 7-H) 8.09 (s, 1H, 3-H), 8.11 (dd, 1H, 5′-H, J3′,5′ 2.9 Hz, J5′,6′ 9.2 Hz), 8.15 (m, 1H, 5-H or 8H), 8.24 (m, 1H, 5-H or 8-H), 8.77 (d, 1H, 3′-H, J3′,5′ 2.9 Hz). 13C NMR (126 MHz): δ 111.28, 115.34, 121.37, 126.03, 126.10, 126.58, 128.03, 128.06, 130.95, 133.18, 133.42, 133.73, 133.92, 137.78, 139.84, 146.07, 149.67, 167.63, 182.93, 183.22. LC-MS (m/z): 501 [M − Na+ + NH4+]+, 484 [M − Na+ + H+]+, 482 [M− Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 95.7%. 3034



Article

P2Y2 receptor was done in analogy (see Supporting Information). Briefly, the coding sequence of the respective receptor was cloned into the pQCXIN or pLXSN retroviral vector, amplified, purified, and sequenced prior to the transfection of GP+env AM-12 packaging cells together with vesicular stomatitis virus G (VSV-G) protein DNA using lipofectamine 2000. After 16 h, 3 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum, 1% of a penicillin/ streptomycin solution (final concentrations: penicillin = 100 U/mL, streptomycin = 0.1 mg/mL), and sodium butyrate (5 mM) was given to the packaging cells and these were kept at 32 °C and 5% CO2 for 48 h, during which the viral vectors containing the receptor sequence were produced and released into the surrounding medium. These were harvested, filtered (45 μm filter pore diameter), and given to 1321N1 astrocytoma cells that do not intrinsically express P2 receptors at a detectable level. Polybrene solution (6 μL, 4 mg/mL in H2O, filtered) was added. After 2.5 h, the virus-containing medium was discarded and DMEM supplemented with 10% fetal calf serum and 1% of a penicillin/streptomycin solution (final concentrations: penicillin = 100 U/mL, streptomycin = 0.1 mg/mL) was given to the cells. These were incubated for 2 days, followed by selection of successfully transfected cells with Geneticin resistance by adding G418 (200 μg/mL) to the medium. Single cells were selected and grown into monoclonal colonies in case of the P2Y2 and P2Y4 receptor expressing cells. Pharmacological Evaluation of the Compounds at Human P2Y1, P2Y2, P2Y4, and P2Y6 Receptors. All experiments were performed using 1321N1 human astrocytoma cells stably expressing the respective human P2Y receptor subtype. The cells were grown in T175 tissue culture flasks (175 cm2 area) containing 25−30 mL DMEM supplemented with 10% fetal calf serum, 200 μg/mL G418, and 1% of a penicillin/streptomycin solution (final concentrations: penicillin = 100 U/mL, streptomycin = 0.1 mg/mL). DMEM and supplements were purchased from Invitrogen (Life Technologies GmbH, Darmstadt, Germany). The flasks were kept at 37 °C in a humidified atmosphere (96% relative humidity) containing 5% CO2. Cells were maintained in the exponential growth phase throughout and regularly tested for mycoplasma contamination. Calcium Mobilization Assays. Calcium measurements were performed as previously described.60 Briefly, 1321N1 human astrocytoma cells stably transfected with the coding sequence for the respective human P2Y receptor were used. Approximately 24 h prior to testing, the nutrient medium was discarded and the cells rinsed with phosphate-buffered saline before detachment using 0.05% trypsin/0.6 mM EDTA. The cells were then suspended in DMEM with the supplements described above and dispensed into sterile, black, flat, clear-bottom 96-well polystyrene microplates with lid (Corning 3340) at 50000 cells per well. The microplates were incubated at 37 °C in humidified air with 5% CO2, during which the cells adhered to the coated bottom of the wells. Test compounds were investigated by measuring their inhibition of P2Y1, P2Y2, P2Y4, or P2Y6 receptormediated intracellular calcium mobilization using a FlexStation 3 (Molecular Devices GmbH, Biberach an der Riss, Germany) plate reader. At the start of the assay, the plated cells were loaded with fluo-4 acetoxymethyl ester (Life Technologies GmbH, Darmstadt, Germany) for 1 h. Excess dye was subsequently removed and Hank’s Balanced Salt Solution (HBSS) buffer given to the cells. Afterward, the cells were preincubated with the test compound for 30 min. Using the pipetting function of the microplate reader, the physiological ligand was injected at a concentration that corresponds to its EC80: 500 nM ADP for the P2Y1, 500 nM UTP for the P2Y2 and P2Y4, and 750 nM UDP for the P2Y6 receptor. The final volume was 200 μL per well. Fluorescence was measured at 525 nm following excitation at 488 nm. At least three independent experiments were performed in duplicate. IC50 values were calculated by nonlinear regression using Prism 5.0 (GraphPad Software, San Diego, CA, USA). Homology Modeling of the Human P2Y4 Receptor. A homology model of the human P2Y4 receptor was created based on the X-ray cocrystal structure of the human P2Y1 receptor with the nucleotide-derived antagonist MRS2500 (PDB-ID: 4XNW, resolution 2.7 Å).56 The structure was downloaded from the RSCB (Research Collaboratory for Structural Bioinformatics) Protein Data Bank

ditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-[(3,4-dimethylphenyl)thio]aniline (137.6 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (7% yield), mp >300 °C. 1H NMR (500 MHz): δ 2.21 (s, 6H, 2CH3), 7.21 (m, 7H, 2′-H, 3′-H, 5′-H, 6′H, 3″-H, 4″-H, 6″-H), 7.84 (m, 2H, 6-H, 7-H), 8.00 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-H), 10.06 (br, 2H, 1-NH2), 11.93 (s, 1H, 4-NH). 13C NMR: δ 19.1, 19.4, 109.4, 112.1, 122.9, 123.6, 126.1, 126.2, 129.3, 130.8, 130.9, 131.1, 131.5, 132.7, 132.9, 133.4, 133.6, 134.2, 136.4, 137.9, 138.5, 140.1, 142.7, 144.6, 182.0, 182.8. LC-MS (m/z): 531 [M − Na+ + H+]+, 529 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 95.6%. Sodium 1-Amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (64). Reaction conditions according to general procedure A: Compound 6a (121.3 mg, 0.3 mmol), 4-[(pyridin-3-ylmethyl)thio]aniline (129.8 mg, 0.6 mmol). MW conditions: 15 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (14% yield), mp >300 °C. 1H NMR (500 MHz): δ 4.33 (s, 2H, CH2), 7.21 (d, 2H, 2′-H, 6′-H), 7.39 (d, 2H, 3′H, 5′-H), 7.64 (m, 1H, 5″-H), 7.84 (m, 2H, 6-H, 7-H), 7.97 (s, 1H, 3H), 8.03 (s, 1H, 6″-H), 8.26 (m, 2H, 5-H, 8-H), 8.60 (2s, 2H, 2″-H, 4″-H), 11.89 (s, 1H, 4-NH). 13C NMR: δ 34.7, 109.4, 112.2, 123.0, 123.4, 125.2, 126.1, 126.2, 129.1, 132.6, 133.0, 133.4, 133.6, 134.3, 136.1, 138.7, 140.2, 140.8, 142.7, 144.6, 145.0, 146.2, 182.0, 182.8. LCMS (m/z): 518 [M − Na+ + H+]+, 516 [M − Na+ + H+]−. Purity by HPLC-UV (254 nm) ESI-MS: 98.6%. 1-Amino-4-(2-carboxy-4-fluorophenylamino)-2-methyl-9,10dioxo-9,10-dihydroanthracene (67). Reaction conditions according to general procedure A: Compound 6b (31.6 mg, 0.1 mmol), 2-amino5-fluorobenzoic acid (31 mg, 0.2 mmol). MW conditions: 5 min, 120 °C, 100 W; pressure up to 10 bar. Analytical data: blue powder (21 % yield), mp > 300 °C. 1H NMR (500 MHz): δ 2.22 (s, 3H, -CH3), 7.02 (m, 1H, 6′-H), 7.17 (m, 1H, 5′-H), 7.50 (m, 2H, 3-H, 3′-H), 7.79 (m, 2H, 6-H, 7-H), 8.24 (m, 2H, 5-H, 8-H). 13C NMR (126 MHz): δ 18.34, 108.74, 111.85, 114.83, 115.02, 116.56, 116.72, 121.77, 121.84, 125.74, 125.86, 126.37, 132.35, 132.49, 133.84, 134.23, 135.24, 135.93, 139.43, 141.12, 146.98, 155.93, 157.82, 168.08, 180.26, 182.27. LC-MS (m/z): 391 [M]+, 389 [M]−. Purity by HPLC-UV (254 nm)-ESI-MS: 99.7 %. 4-(3-Fluorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2sulfonic Acid (68). Reaction conditions: Compound 12 (41 mg, 0.1 mmol) was reacted according to general procedure C. Analytical data: reddish-purple powder (97% yield), mp >300 °C. 1H NMR (600 MHz): δ 7.07 (td, 1H, 6′-H), 7.23 (m, 2H, 2′-H, 5′-H), 7.51 (m, 1H, 4′-H), 7.80 (d, 1H, 1-H or 3-H, J1,3 1.47 Hz), 7.86 (d, 1H, 1-H or 3-H, J1,3 1.47 Hz), 7.90 (td, 1H, 6-H or 7-H), 7.94 (td, 1H, 6-H or 7-H), 8.20 (dd, 1H, 5-H or 8-H), 8.25 (dd, 1H, 5-H or 8-H), 11.21 (s, 1H, 4NH). 13C NMR (151 MHz) δ 39.52, 110.16, 110.32, 111.46, 111.60, 113.94, 115.53, 116.05, 119.18, 119.20, 126.51, 126.66, 131.26, 131.33, 132.49, 133.98, 134.17, 134.30, 134.68, 140.94, 141.01, 147.72, 154.35, 161.98, 163.60, 182.30, 184.50. LC-MS (m/z): 415 [M − H+ + NH4+]+, 398 [M]+, 396 [M]−. Purity by HPLC-UV (220−650 nm) ESI-MS: 98.5%. 4-(3-Methoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene2-sulfonic Acid (69). Reaction conditions: Compound 27 (42 mg, 0.1 mmol) was reacted according to general procedure C. Analytical data: reddish-purple powder (38% yield), mp >300 °C. 1H NMR (500 MHz) δ 3.80 (s, 1H, −OCH3), 6.84 (m, 1H, 6′-H), 6.94 (m, 2H, 2′-H, 4′-H), 7.39 (m, 1H, 5′-H), 7.81 (d, 1H, 1-H or 3-H, J1,3 1.5 Hz), 7.83 (d, 1H, 1-H or 3-H, J1,3 1.5 Hz), 7.90 (td, 1H, 6-H or 7-H), 7.95 (td, 1H, 6-H or 7-H), 8.20 (dd, 1H, 5-H or 8-H), 8.27 (dd, 1H, 5-H or 8H), 11.24 (s, 1H, 4-NH). 13C NMR (126 MHz) δ 55.20, 109.27, 110.86, 113.28, 115.08, 115.70, 116.14, 126.46, 126.61, 130.47, 132.51, 133.84, 134.15, 134.25, 134.61, 140.03, 148.47, 154.31, 160.36, 182.35, 184.35. LC-MS (m/z): 427 [M − H+ + NH4+]+, 410 [M]+, 408 [M]−. Purity by HPLC-UV (220−600 nm) ESI-MS: 95.2%. Retroviral Transfection of 1321N1 Astrocytoma Cells with Human P2Y1, P2Y4, and P2Y6 Receptors. Transfection of 1321N1 astrocytoma cells with human P2Y1, P2Y4, and P2Y6 receptors was performed as previously described.59 Transfection with the human 3035



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(http://www.rcsb.org/) and used as a template for generating the homology model of the human P2Y4 receptor.61 The amino acid sequence of the human P2Y4 receptor with the accession number P51582 was retrieved from UniProtKB sequence database.62 The sequences of the human P2Y1 and P2Y4 receptors were aligned using Clustal Omega and AlignMe (Supporting Information, Figure 58).63,64 The resulting alignments were manually adjusted for improving the alignment, particularly in the transmembrane and extracellular loop region 2 (ECL2). An overall sequence identity of 41.8% and a similarity of 60.2% between the human P2Y1 and P2Y4 receptors was calculated. The resulting alignment was used as input for generating homology models of the human P2Y4 receptor using Modeller9.16.65,66 From the 750 models generated, the best model was selected on the basis of the Discrete Optimized Protein Energy (DOPE) score included in Modeller, manual visualization of the presence of an ionic lock between Asp187 and Arg292, and analysis of the binding sites. The overall structural quality was confirmed by a Ramachandran plot (Supporting Information, Figure 59),67 and sequence−structure compatibility of the model was ensured using PROSA II profile analysis (Supporting Information, Figure 60).68 Possible binding sites were identified using the SiteFinder module from Molecular Operating Environment (MOE 2014.09).69 Docking Studies. Docking simulations were performed using Induced Fit Docking (IFD) and Glide as implemented in Schrödinger release 2016.70−72 Prior to docking, the homology model of the human P2Y4 receptor was prepared using the Protein Preparation Wizard at pH 7.4 and with force field Optimized Potentials for Liquid Simulations Version 3 (OPLS3) implemented in Schrödinger. The ligands were prepared using the implemented LigPrep module and the OLPS3 force field in possible states at pH 7.4 ± 1.0. The conformations of the docked ligands within an energy window of 2.5 kcal/mol were considered. For Glide docking, the following standard parameters were selected: receptor van der Waals scaling, 0.50; ligand van der Waals scaling, 0.50; a maximum of 20 poses per ligand. Residues within 5.0 Å of the ligand poses were refined, and the side chains were optimized. The best docking pose was selected based on the IFD score and Prime Energy values. The compounds were subsequently docked to the best scoring complex obtained for compound 61. A receptor grid center was specified on the basis of the transformed position of the allosteric antagonist MRS2500 from the human P2Y1 receptor structure, with a cubic grid side length of 10 Å. As precision setting, XP (extra precision) was chosen. Following Glide docking, the resulting poses were selected using the IFD scores and Prime Energy as representative values.

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Christa E. Müller: 0000-0002-0013-6624 Younis Baqi: 0000-0002-9659-8419 Author Contributions §

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Y.B. is grateful for an SQU grant (SR/SCI/CHEM/15/01). E.M.M. thanks The Deutscher Akademischer Austauschdienst (DAAD) for a Ph.D. scholarship. M.R. and C.E.M. are grateful to the NRW International Graduate Research School BiotechPharma for financial support. A.N. and C.E.M. were supported by the Deutsche Forschungsgemeinschaft (DFG, Research Training group GRK 1873). We thank Schrödinger Inc., for providing the evaluation license.

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ABBREVIATIONS USED 2MeSADP, 2-methylthioadenosine-5′-diphosphate; APP, amyloid precursor protein; DMEM, Dulbecco’s Modified Eagle’s Medium; ECL, extracellular loop; GIT, gastrointestinal tract; IFD, induced fit docking; MRS2500, 2-iodo-N6-methyl-(N)methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid; RB-2, reactive blue 2; SEM, standard error of the mean; TM, transmembrane region; UTP, uridine-5′-triphosphate

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00030. LC-MS spectra of RB-2 P (2′); 1H NMR, 13C NMR, and DEPT 135 NMR spectra of the newly synthesized anthraquinone derivatives; description of the β-arrestin recruitment assay used for measuring P2Y12 receptor antagonism; multiple sequence alignment of P2Y receptor subtypes (PDF) Molecular formula strings (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For C.E.M.: phone, +49-228-73-2301; fax, +49-228-73-2567; E-mail, [email protected]. *For Y.B.: phone, +968-24141473; fax, +968-24141469; E-mail, [email protected]. ORCID

Enas M. Malik: 0000-0003-4563-3584 3036



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